Overexpression and characterization of two types of nitrile hydratases from Rhodococcus rhodochrous J1.

Yao Lan,Li Zhou,Zhemin Zhou,Wenjing Cui,Zhongmei Liu,Ruihua Shen,Xianping Zhong,Xiaohuan Zhang,Guo-Qiang Chen

doi:10.1371/journal.pone.0179833

Abstract

Nitrile hydratase (NHase) from Rhodococcus rhodochrous J1 is widely used for industrial production of acrylamide and nicotinamide. However, the two types of NHases (L-NHase and H-NHase) from R. rhodochrous J1 were only slightly expressed in E. coli by routine methods, which limits the comprehensive and systematic characterization of the enzyme properties. We successfully expressed the two types of recombinant NHases in E. coli by codon-optimization, engineering of Ribosome Binding Site (RBS) and spacer sequences. The specific activity of the purified L-NHase and H-NHase were 400 U/mg and 234 U/mg, respectively. The molecular mass of L-NHase and H-NHase was identified to be 94 kDa and 504 kDa, respectively, indicating that the quaternary structure of the two types of NHases was the same as those in R. rhodochrous J1. H-NHase exhibited higher substrate and product tolerance than L-NHase. Moreover, higher activity and shorter culture time were achieved in recombinant E. coli, and the whole cell catalyst of recombinant E. coli harboring H-NHase has equivalent efficiency in tolerance to the high-concentration product relative to that in R. rhodochrous J1. These results indicate that biotransformation of nitrile by R. rhodochrous J1 represents a potential alternative to NHase-producing E. coli.

Highlights

Nitrile hydratase (NHase, EC 4.2.1.84) catalyzes the hydration of a nitrile molecule to the corresponding amide [1]
To heterologously express lowmolecular-weight NHase (L-NHase) in E. coli, the gene cluster of nhlBAE were inserted into pET24a resulting in the recombinant plasmid pET24a-nhlBAE
E. coli transformant harboring pET24a-nhlBAE was used for expression of L-NHase

Summary

Introduction

Nitrile hydratase (NHase, EC 4.2.1.84) catalyzes the hydration of a nitrile molecule to the corresponding amide [1]. NHase forms a hetero-tetramer that is composed of α- and β-subunits with either a non-heme iron (Fe-NHase) or non-corrin cobalt ion (Co-NHase) in the active center [2]. The α-subunits, which share a characteristic metal-binding motif [CXLC(SO2H)SC (SOH)] containing two modified cysteine residues, cysteine-sulfinic acid (αCys-SO2H) and cysteine-sulfenic acid (αCys-SOH), bind the metal ions in both Co-NHase and Fe-NHase. International S&T Innovation Cooperation Key Project, 2016YFE0127400, www.most.gov.cn/ ZL. Fundamental Research Funds for the Central Universities JUSRP51713B www.moe.gov.cn/ WC. The funder provided support in the form of research materials, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The company Rudong Nantian Nongke Chemical Engineering Co. Ltd and ENNova Health Science Technology Co., Ltd., ENN Group participated in the data analysis and a part of fermentation experiment

Methods

Results

Conclusion